The siRNA (small interfering RNA) has been widely used as a method to repress expression of a desired target gene through RNA interference, but it also causes non-specific repression of other genes, off target effects, as an inevitable disadvantage, raising the serious concern of leading to faulty research results or side effects in therapeutic treatments. The off-target effects are occurred since Argonaute protein, the core effector in RNA interference, treats siRNA, which is artificially introduced in order to induce RNA interference, as a miRNA (microRNA) existing in a cell. Therefore, it is called miRNA-like off-target effect. The miRNA recognizes a target gene majorly through base-pairing with a seed region (positions 2-7 from the 5′ end) for suppression, and the off-targets caused by siRNAs are also induced depending on sequences of the seed regions as well. The miRNA-like off-target effects in siRNAs have been already reported in several studies (Jackson, A. L., et al., Nat. Biotechnol., 21(6): 635, 2003; Jackson, A. L., et al., Rna, 12(7): 1179, 2006; Birmingham et al., Nat. Methods, 3(3): 199, 2006; Lin et al., Nucleic Acids Res., 33(14): 4527, 2005; Anderson et al., RNA, 14(5): 853, 2008), and affect expression of at least hundreds and at most ˜1500 of genes depending on sequences of the seed regions and are serious enough to cause up to 30% of the positive hits in siRNA based phenotype screening. Additionally, in the case of miRNAs, they are also reported to silence target genes through compensatory pairings within their 3′ end regions (3′-compensatory pairing) when the interactions between seed regions and targets become weak (Cell. 2009; 136:215-233), implicating that the miRNA-like off-target effects are likely to be mediated by such mechanism.
In addition, due to such widespread off-target silencing effects mediated by siRNAs, several chemical and structural modifications have been attempted to reduce the off-target silencing while maintaining the efficiency of suppressing an intended target. A modification adding methyl groups to a 2′ position of a ribosyl ring of the nucleotide (2′OMe) was studied and used by Dharmacon Research (Lafayette, Colo.) to suppress the off-target effects, initially found to be effective in reducing both the number of off-targets and the extent of off-target effects especially when the 2′OMe is at a position 2 from the 5′ end region, but the silencing of the intended target gene is also somewhat reduced. Since then, another kinds of modifications such as LNA modification (Puri et al., Nucleic Acids Symp. Ser 0.2008), UNA modification (Bramsen et al., Nucleic Acids Res. 2010; 38, 5761-73), and bulge modification introducing a single nucleotide bulge (Mol Ther. 2011 September; 19(9):1676-87) also have been developed.
However, all such chemical modifications were applied to a nucleotide backbone rather than to a base of which sequence is critical to cause off-targets, unable to affect a fundamental base-pairing. Because of this reason, they cannot completely block off-target effect albeit somewhat can be reduced, also having a problem to reduce efficiency of on-target silencing.